Virus preparations and methods

ABSTRACT

Methods are disclosed for the preparation of herpesvirus, such as herpes simplex virus type 2 for vaccine use. Such viruses can be grown on serum free or serum containing media and can be prepared from the virus containing culture supernatant or virus containing cells. The virus is prepared for subsequent pharmaceutical formulation by methods which may include treatment with solid phase affinity reagents containing sulfate- or sulfonate-comprising binding groups. Such sulfated polysaccharide groups as heparin or dextran sulfate may be used, and eluted with salt solutions. The process can be combined with other culture, harvesting and formulation steps.

CROSS-REFERENCE TO RELATED APPLICATIONS

Not Applicable.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

Not applicable.

INCORPORATION BY REFERENCE OF MATERIAL SUBMITTED ON A COMPACT DISC

Not Applicable.

BACKGROUND OF THE INVENTION

(1) Field of the Invention

This invention relates to the production, the harvesting and thepurification of virus from virus-infected cell cultures, for example forexperimental and therapeutic purposes, e.g. for the production ofpharmaceutical formulations such as prophylactic or therapeuticinoculations. In particular aspects, the invention relates to methodsand arrangements for the production of preparations of herpesviruses.Other aspects of the invention will be apparent from the descriptiongiven below.

(2) Description of the Related Art Including Information Disclosed Under37 CFR 1.97 and 37 CFR 1.98.

Several methods are known for producing live virus preparations all ofwhich include extraction of the virus from a virus infected cell, e.g.Vero cells or MRC5 cells among others, for vaccine, therapeutic andother purposes.

U.S. Pat. No. 3,985,615 (Osaka Research Foundation: T Kubo et al) showsproduction of live attenuated varicella virus for vaccine use by culturecomprising passage in guinea pig primary embryonic tissue cells.

U.S. Pat. No. 5,024,836 (Merck: W J McAleer et al) relates to productionof lyophilized vaccine preparations based thereon. Also disclosed is thefact that aqueous solutions of live virus vaccines are known to beunstable during storage.

DD-209738 (Cent Cerc Bioprep: IV Patrascu) illustrates production ofanother type of herpesvirus, for use as vaccine against Marek's disease.The herpesvirus is produced by (a) culturing specific-pathogen-freechicken embryo cells on dextran microspheres; (b) inoculating theculture at 80% confluence with turkey herpes virus strain FC-126 (clone1, IIIb); (c) collecting the infected cells in SPGA medium (sucrose,phosphate, glutamate, bovine albumin fraction V) when the cytopathiceffect is 80%; (d) subjecting the suspension to three ultrasonic pulsesof 1 minute duration at 2 minute intervals and centrifuging it torecover a first crop of vaccine; (e) resuspending the sediment in SPCAmedium and repeating step (d) to obtain a second crop of vaccine (toincrease the vaccine yield by almost 2%); (f) freezing the combinedvaccines at −100° C. prior to determining the virus titer; and (g)diluting the SPCA medium and freeze drying.

JP06234659-A (Z H Handai Biseibutsubyo Kenkyukai) describes, in anexample, production of herpesviral vaccine on human diploid fibroblastMRC-5 cells cultured in MEM medium at 37° C., comprising inoculation ofvaricella virus Oka strain seed virus at a MOI of 0.03 to MRC-5 cellsand culture at 37° C. for 2 days. Virus is then suspended in a solutioncontaining 6.4 g NaCl, 0.16 g KCl, 2.3 g Na₂HPO₄ 12H₂O, 0.16 g KH₂PO₄,50.0 g sucrose, 1.0 g Na L-glutamate, 2.0 g gelatin, 25.0 g gelatinhydrolysate and 0.1 g EDA-3Na per L.

EP 0 573 107, U.S. Pat. No. 5,360,736 and U.S. Pat. No. 5,607,852(Merck: P A Friedman et al) describe processes for production ofattenuated varicella zoster virus vaccine, including a process forpreparing live, attenuated, cell-free varicella-zoster virus (VZV)vaccine that comprises: (a) Culturing VZV infection-susceptible cells,selected from human diploid cells, to confluency in monolayer culture,under conditions of sufficiently high nutrition to achieve a high degreeof cell replication, and supplying a non-metabolizable disaccharide; (b)infecting the cells cultured according to step (a) at as close to thepoint of confluency as possible with as high a multiplicity of infectionof VZV-infected cells as practical; (c) maintaining the VZV-infectedculture in a state of high nutrition for about 22-96 hours andharvesting at the point of peak infectious VZV production; (d) washingthe VZV-infected culture with a physiologic solution, optionallycontaining a lysosomotropic agent, such as ammonium chloride orchloroquine, prior to harvesting the VZV infected cells; (e) Harvestingthe VZV infected cells into a minimal volume of a stabilizing solutionand either disrupting the cells immediately or freezing the cells forlater disruption, (1) Disrupting the VZV-infected cells to optimallyrelease cell-associated VZV, and removing cellular debris, to provide acell-free VZV preparation. The process is proposed for mass productionof live vaccine. Appropriate nutrient medium for growing cells inmonolayer culture in that connection is described as consistingessentially of SRFE-2 medium supplemented with between 0.2 mg/mL and 0.4mg/mL soybean lipid and 10% fetal calf serum, the cells being selectedfrom MRC-5 cells, WI-38 cells and Vero cells.

U.S. Pat. No. 5,665,362 (Cantab Pharmaceuticals Research: S C Inglis etal) and U.S. Pat. No. 5,837,261 (Cantab Pharmaceuticals Research: SCInglis et al) disclose recombinant cells and culture methods forproducing genetically disabled herpesvirus such as herpes simplex virusfor vaccine purposes, wherein the virus is grown on complimentary cells.

U.S. Pat. No. 6,267,967 (Cantab: M D Johnston et al) describe processesfor purification of herpes simplex virus. Infectious preparations ofhuman herpesviruses such as herpes simplex virus (HSV), e.g. HSV type 2(HSV-2), which tend to remain strongly cell-associated when grown inculture and affinity reagent carrying the virus, which can be appliedfrom a carrier liquid containing salt (e.g. sodium chloride or otherpharmaceutically acceptable salt over about 0.4M) or containing heparinor another sulfated or sulfonated polysaccharide (e.g. in the order ofabout 10-250, such as about 50, micro-g/mL), can then suitably be washedand the virus recovered in actively infectious form by elution, e.g.with high-concentration salt solution or with sulfated or sulfonatedpolysaccharide. The initial harvesting of virus from such a cell culturecan be carried out in any of a variety of ways. Examples of methodsinclude cell rupture, e.g. by freeze-thaw cycles or osmotic stressprocedures, e.g. with hypotonic saline or glycerol solutions,sonication, elution by heparin or dextran sulfate or equivalent, or byusing elution with saline solution.

U.S. Pat. No. 6,013,265 (UMB: L Aurelian), incorporated herein byreference, or U.S. Pat. No. 6,054,131 (UMB: L Aurelian), incorporatedherein by reference, disclose a growth compromised herpes simplexrecombinant virus, e.g. HSV-2, in which the PK domain has been deleted.In a wild type HSV-2 virus, replication began at 2 hours post-infectionand reached peak levels at 36 hrs post-infection. In the PK deletedHSV-2, the onset of replication was not seen until 15 hrs post-infectionboth in 10% serum and 0.5% serum supplemented Vero cells. Whenreplication resumed this mutant reached titers similar to those of HSV-2at 36 hrs post infection in the presence of 10% serum, but not in cellssupplemented with in 0.5% serum in the medium.

It remains desirable to provide methods for production ofherpesvirus-containing preparations, capable of contributing to themanufacture of infectious virus preparations in good yield and purity,e.g. those that are to be used in vaccines or as therapies.

BRIEF SUMMARY OF THE INVENTION

Embodiments of the invention include processes for preparing herpesvirusfor a vaccine or therapy, which comprise (a) culturing host cellsinfected with the virus, and (b) separating of the medium from theinfected cells and (3) using the supernatant to provide a viruspreparation suitable for immunotherapy or vaccination of an animal.

It is, accordingly, an objective of embodiments of the present inventionto provide processes for the isolation of herpesvirus for use in avaccine or therapy.

Another objective of embodiments of the present invention to provideprocesses for the isolation of herpesvirus which are simple,inexpensive, and easy to implement.

Another objective of embodiments of the present invention to provideprocesses for the isolation of herpesvirus which do not involve theaddition of additives to aid in purification.

Another objective of embodiments of the present invention to provideprocesses for the isolation of herpesvirus which do not require thedisruption of cells used to produce the herpesvirus.

Another objective of embodiments of the present invention to provideprocesses for the isolation of herpesvirus which utilize onlyinexpensive components.

Another objective of embodiments of the present invention to provideprocesses for the growth of herpesvirus which are performed in theabsence of serum.

Another objective of embodiments of the present invention to provideprocesses for the isolation of herpesvirus which are performed in theabsence of serum

A final objective of embodiments of the present invention to provideprocesses for the isolation of herpesvirus which involve easy to obtainand inexpensive reagents and are without adverse effects on theenvironment.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING

Not Applicable.

DETAILED DESCRIPTION OF THE INVENTION

We have found, most surprisingly, that an HSV-2 which has been deletedin the PK domain is readily transferred beyond the cell wall of the cellwherein it has been grown and is found in abundance in the culture fluidas well as in the cell. Virus levels in each area are found to be within1 log of the levels of the other and since the supernatant contains30-300 times, more or less, volume than does the cell mass, theavailable level of virus is substantially higher in the supernatant thanin the cell. This obviates the need for cell rupture to free the virusparticle found normally within the infected cell. Cell rupture has thedisadvantages of requiring extensive purification of impuritiesconsisting of cellular components and of reagents added to effect cellrupture.

Again, most surprisingly, we have found that cellular media which isserum free is compatible with growth of an HSV-2 which has been deletedin the PK domain, even though this mutant had previously been shown tobe growth compromised in medium containing 10% fetal calf serum and notreproductive in medium containing 0.5% fetal calf serum.

In embodiments of the invention, suitable media for virus productioninclude, for example, EMEM with 10% serum, or for example, VP-SFM(Invitrogen, Bethesda, Md.) which has a very low protein concentration(5 μg/mL and contains no proteins or peptides of animal or human origin)but does contain human recombinant epidermal growth factor (EGF) andrecombinant human insulin or, as an example, RenCyte (In Vitro Servicesand Systems, Göttingen, Germany), which has no protein or serum. Othercommercially available media that could be used, for example, includePC-1 or HL-1 (Cambrex Bioscience, Walkersville, Md.) which have addedinsulin.

In embodiments of the invention, cell lines which support the limitedgrowth of PK deleted recombinant HSV-2 include cells mentioned in U.S.Pat. No. 6,013,265 (UMB: L Aurelian) or U.S. Pat. No. 6,054,131 (UMB: LAurelian) and most preferably are Vero or MRC-5 cells.

In embodiments of the invention, a much higher degree of purity isobtained by use of the cell supernatant as opposed to rupturing thecells, since contaminating proteins are retained within the cell, andICP10deltaPK allows a very high concentration of viral particles in thecell growth medium, e.g. up to 10⁹ pfu/mL.

In embodiments of the invention affinity purification can typically becarried out on a virus supernatant that has been obtained from a cultureof suitably infected host cells such as Vero cells or MRC-5. It can beconvenient to pass such an initially-harvested viral preparation througha membrane filter, e.g. on approximately 5 micron or finer membranefilter, to yield a clarified viral suspension, before the affinitypurification. Using examples of the invention e.g. as described below,it is possible to prepare viral fractions containing usefully reducedlevels of DNA and protein relative to the virus titer. The viral productof the affinity purification can if desired be subjected to any furtherchosen purification steps. The virus preparation harvested in this waycan be treated with nuclease enzyme either before (or less preferablyafter) the affinity purification, to reduce any content of contaminatingnucleic acid to acceptable levels. The virus preparations obtained bythe use of processing steps as described herein can be further processedand made part of pharmaceutical compositions, e.g. with per-seconventional ingredients of virus vaccines or other pharmaceuticallyacceptable agents useful in stabilizing herpevirus. The invention isfurther described and illustrated by the following non-limitativeexample.

EXAMPLE 1

A process according to an example of the invention, for harvesting andpurifying virus particles, can make use of a culture of Vero cellsinfected with HSV-2 (e.g. a PK deleted mutant of HSV2 as described inU.S. Pat. No. 6,013,265 (UMB: L Aurelian) or U.S. Pat. No. 6,054,131(UMB: L Aurelian) for vaccine use), and grown essentially in knownmanner in 10% serum with Eagle's minimum essential medium (EMEM)supplemented with 10% fetal calf serum (FCS) and antibiotics. ConfluentVero cells were infected with HSV-2, ICP10deltaPK (CS) at a multiplicityof infection of about 0.01 and incubated at about 34° C. When cytopathiceffect is observed to be 80-100%, e.g. 24-72 hours after infection, theculture can be treated as ready for virus harvest.

Alternatively, the Vero cells can be infected with HSV-2 and grown asdirected in a serum free medium such as VP SFM from Invitrogen,Bethesda, Md. or RenCyte (In Vitro Services and Systems, Göttingen,Germany).

To harvest the virus, the culture medium is decanted and used directlyor purified further. Levels of virus using RenCyte or VP-SFM are alsosuitable for use. The medium containing the virus can then be useddirectly to immunize for pharmaceutical use, stabilized withstabilization agents or may be further purified.

For example, pre-filtration can be carried out e.g. with a filter havinga pore size in the range from 0.4-5 micron (not critical) to yield aclarified viral suspension, before further affinity purification. Thesupernatant liquid from centrifugation can be diluted or diafiltered toobtain suitable ion concentrations.

The culture medium may optionally be treated with a nuclease enzyme(Benzonase™ which has DNase activity, and RNase activity) at up to about50 units/mL in the presence of about 2-10 mM magnesium ion, e.g. for upto about 1 hour at a temperature from about 4° C. up to roomtemperature. However, it can often be found that the culture medium hasa sufficiently low content of DNA that a separate DNase treatment stepis unnecessary.

The virus-containing liquid may be further purified on a Pharmaciaheparin HP column chromatography material (based on a highlycross-linked agarose gel) (e.g. of diameter about 34 micron) obtainablefrom Pharmacia Biotech in the form of HiTrap™ prepared columns.Alternatively, the affinity reagent can be beads of Fractogel™ FMD SO₅650 M from Merck (Darmstadt) as described above, used in generallysimilar manner.

In a further example of this step, a HSV-2 virus culture supernatantcould be applied to a heparin column prepared by washing with phosphatebuffered saline. Approximately 100 mL of virus preparation containing asmuch as 10⁸ pfu/mL might be loaded on to the column. The virus could befractionally eluted and the fractions containing the concentrated viruscould be collected.

If desired, as an optional further purification step, the intermediatevirus-containing eluate from the column could be subjected to tangentialcross-flow filtration (diafiltration) e.g. using a filter/membrane witha 500 kD exclusion limit in a Filtron™ or other tangential crossflowdevice.

The retentate from the cross-flow ultrafiltration step could optionally,if desired, be treated by diafiltration against citrate/saline buffer,and the retentate finally subjected to 0.2 micron (sterilizing)filtration optionally preceded by filtration with a filter of from about0.45 micron to 5 micron, using the same buffer again.

If desired, this step can be used to make the liquid containing thevirus preparation up to about 20 mg/mL in a suitable stabilizer such asa stabilizing protein, e.g. human serum albumin at about 20 mg/mL. Itcan sometimes be useful to prewash the filters with a liquid containingthe same stabilizer in the same buffer, before using the filters totreat the virus preparation.

The resulting product can be obtained as a suspension of virus particlesin a stabilizer, in which the level of residual DNA can besatisfactorily low.

The invention can be usefully applied, for example to the culture andharvesting of a recombinant HSV-2 virus for vaccine use, which virus hasa deletion in respect to the PK gene and has been cultured on a cellline of Vero cells or MRC 5 cells.

The present invention and disclosure extend to the methods andcompositions and the resulting products as described herein, and tomodifications and variations of the steps and features mentioned anddescribed in the present description and claims, including allcombinations and subcombinations of the steps and features hereof,including variations in the order and selection of steps, and thedocuments cited herein are hereby incorporated by reference in theirentirety for all purposes.

1. A process for preparing herpesvirus for a vaccine or therapy, whichcomprises: (a) culturing host cells infected with the virus, (b)separating of the medium from the infected cells and (c) using thesupernatant to provide a virus preparation suitable for immunotherapy orvaccination of an animal.
 2. The process of claim 1 wherein saidherpesvirus is an HSV-2 which has the PK domain deleted.
 3. The processof claim 1 wherein said herpesvirus is ICP10deltaPK.
 4. The process ofclaim 1 wherein said culture is carried out in a serum free medium. 5.The process of claim 1 wherein said culture is carried out in a mediumcontaining low levels of protein.
 6. The process of claim 1 where saidculture is carried out in a medium with added epidermal growth factor.7. The process of claim 1 where said culture is carried out in a mediumwith added insulin.
 8. The process of claim 1 further comprising: (d)contacting the herpesvirus-containing preparation to be purified, withan affinity binding reagent that comprises a solid phase carrying asulfate or sulfonate-comprising binding group that can bind materialswith affinity for heparin, thereby to bind said herpesvirus to theaffinity binding reagent, and (e) eluting said herpesvirus from saidbinding reagent.
 9. A process according to claim 1, further comprisingthe step: (d) formulating said herpesvirus with a pharmaceuticallyacceptable carrier, excipient or stabilizing agents.
 10. A processaccording to claim 9 further comprising the step: (e) sterilizing andfreezing or freeze-drying the preparation.
 11. A process according toclaim 1 wherein the herpesvirus comprises infectious herpesvirus.
 12. Aprocess according to claim 1 wherein the resulting pharmaceuticalformulation is for use as an immunotherapy.
 13. A process according toclaim 8, wherein said affinity binding reagent carries binding groupscontaining sulfate and nonionic polar groups.
 14. A process according toclaim 8, wherein said affinity binding reagent carries heparin ordextran sulfate.
 15. A process according to claim 8, wherein saidaffinity binding reagent carries sulfated polysaccharide groups.
 16. Aprocess according to claim 8, wherein said affinity binding reagentcarries pendent polyacrylamide chains substituted by sulfoisobutylgroups.
 17. A process for growing herpesvirus comprising the step: (a)culturing host cells infected with the virus in a serum free medium. 18.The process of claim 17 wherein the herpesvirus is an HSV-2 which hasthe PK domain deleted.